RESUMO
The Roseolovirus genus of the Betaherpesvirinae consists of the very closely related viruses, human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) plus the somewhat more distantly related human herpesvirus 7 (HHV-7). The roseoloviruses each encode a homolog of the alphaherpesvirus origin binding protein (OBP) which is required for lytic DNA replication. In contrast, members of the other betaherpesvirus genera, the cytomegaloviruses, initiate DNA replication by a different mechanism. To better understand the basis of roseolovirus OBP sequence specificity, we investigated their ability to recognize each other's binding sites. HHV-6A OBP (OBP(H6A)) and HHV-6B OBP (OBP(H6B)) each bind to both of the HHV-7 OBP sites (OBP-1 and OBP-2) with similar strengths, which are also similar to their nearly equivalent interactions with their own sites. In contrast, HHV-7 OBP (OBP(H7)) had a gradient of binding preferences: HHV-7 OBP-2 > HHV-6 OBP-2 > HHV-7 OBP-1 > HHV-6 OBP-1. Thus, the roseolovirus OBPs are not equally reciprocal in their recognition of each other's OBP sites, suggesting that the sequence requirements for the interaction of OBPH7 at the OBP sites in its cognate oriLyt differ from those of OBPH6A and OBPH6B.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Roseolovirus/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Roseolovirus/classificação , Roseolovirus/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).
Assuntos
Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 7 , Origem de Replicação/genética , Proteínas Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Sequência Consenso/genética , DNA Viral/química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/metabolismo , Análise Heteroduplex , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Viral/análise , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genéticaRESUMO
Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B, respectively) encode homologs (U94) of the parvovirus nonstructural gene, ns1 or rep. Here we describe the HHV-6B homolog and analyze its genetic heterogeneity and transcription. U94 nucleotide and amino acid sequences differ by approximately 3.5% and 2.5%, respectively, between HHV-6A and HHV-6B. Among a collection of 17 clinically and geographically disparate HHV-6 isolates, intravariant nucleotide and amino acid sequence divergence was less than 0.6% and 0.2%, respectively; all 13 HHV-6B isolates had identical amino acid sequences. The U94 transcript is spliced to remove a 2.6-kb intron and is expressed at very low levels relative to other HHV-6B genes, reaching approximately 10 copies per cell 3 days after infection. The mRNA has several small AUG-initiated open reading frames upstream of the U94 open reading frame, a hallmark of proteins expressed at low levels. Consistent with this, the U94-encoded protein was immunologically undetectable in HHV-6B-infected cells. The high degree of sequence conservation suggests that the gene function is nearly intolerant of sequence variation. The low abundance of U94 transcripts and the presence of encoded inefficient translation initiation suggest that the U94 protein may be required only in small amounts during infection.
